Environmentally sustainable synthesis of whey-based carbon dots for ferric ion detection in human serum and water samples: evaluating the greenness of the method

Carbon dots (CDs) are valued for their biocompatibility, easy fabrication, and distinct optical characteristics. The current study examines using whey to fabricate CDs using the hydrothermal method. When stimulated at 350 nm, the synthetic CDs emitted blue light at 423 nm and revealed a selective response to ferric ion (Fe3+) in actual samples with great sensitivity, making them a suitable probe for assessing Fe3+ ions. The produced carbon dots demonstrated great photostability, high sensitivity, and outstanding biocompatibility. The findings showed that Fe3+ ions could be quickly, sensitively, and extremely selectively detected in an aqueous solution of carbon dots, with a revealing limit of 0.409 μM in the linear range of 0–180 μM. Interestingly, this recognition boundary is far inferior to the WHO-recommended threshold of 0.77 μM. Two metric tools which were AGREE and the ComplexGAPI were also used to evaluate the method's greenness. The evaluation confirmed its superior environmental friendliness.


Introduction
CDs, or luminescent nanoparticles, have garnered widespread attention in recent years.2][3][4] Moreover, they possess little toxicity, 5 outstanding photostability, 6 and high water solubility. 7There are two main synthetic approaches for CDs: the topdown and bottom-up methods, which are categorized based on the relationship between sources and products. 8The top-down method involves the utilization of carbon-rich materials or molecules as carbon sources, cutting larger carbon structures like graphite, carbon nanotubes, carbon soot, activated carbon, and graphite oxide into smaller CDs through techniques such as arc discharge, 9 laser ablation 10 and electrochemical methods. 11On the other hand, bottom-up methods use organic molecules, such as glucose 12 and fructose 13 to react and form larger CDs by applying external energy like ultrasonication, 14 microwave pyrolysis, 15 heating, 16 hydrothermal, 17 and microwave irradiation. 18Due to their unique properties, CDs have found applications in various elds, replacing toxic semiconductor quantum dots 19 made of heavy metals like cadmium 20 and lead. 21CDs are used in sensing, 22 electronics, 23 photocatalysis, 24 and biomedical applications. 25,26hey can be synthesized from diverse precursors, including wastes, edibles, and chemical substances, 27 such as watermelon peel, 28 mango, 29 garlic, 30 and ethanol. 31CDs have been applied in imaging mycobacterium and fungal cells, using carbon dots derived from apple juice, 32 and in drug delivery and gene transfer. 33Additionally, nitrogen-doped carbon dots demonstrated by Liang served as an "on-off-on" uorescent sensor for Fe 3+ and glutathione detection. 34n this study, we developed a method for synthesizing CDs from yogurt, a widely popular fermented food enjoyed globally.Renowned for its delightful taste, yogurt frequently features on lists of healthy foods due to its rich content of vitamins, minerals, and proteins, a known source of nitrogen atoms that enhance the quantum efficiency of CDs. 35Moreover, yogurt is affordable and widely accessible in most supermarkets and convenience stores.This marks the pioneering synthesis of CDs from fermented milk water using the hydrothermal method.The characterization of the CDs was conducted with great care using UV-vis, uorescence, FTIR, XRD spectroscopy, DLS, and TEM.
These CDs found application in various sensing scenarios, particularly in the detection of the metal ion Fe 3+ in real samples.Our developed uorescence sensor demonstrated excellent accuracy and stability in determining Fe 3+ levels in drinking water and blood samples.Furthermore, CDs exhibited a selective response to Fe 3+ even in complex biological environments.
Metal ions play a crucial role in regulating the health of the human body, inuencing various biological processes, and serving as essential elements for enzymatic reactions. 36,37mong these, iron stands out as a vital metal ion, contributing to normal biological functions alongside other essential ions like sodium, calcium, zinc, magnesium, and cobalt.Notably, iron functions as a central component in proteins such as hemoglobin, the blood protein. 38Due to its involvement in electron transfer and essential biological processes like cellular respiration, energy production, and oxygen transport, [39][40][41] iron plays a pivotal role in enzymatic reactions.][44][45] Maintaining the proper concentration of iron is crucial for sustaining the body's functions.
Water, a fundamental resource for human and environmental well-being, faces signicant threats from pollution.Major contributors to water pollution include the discharge of domestic sewage, industrial wastes (from sectors like textile, leather, and dyeing), radioactive wastes, as well as fertilizers and pesticides from agricultural elds.The accumulation of industrial wastes and heavy metals in water bodies poses serious threats to both human and animal life. 46Therefore, there's a need for simple, effective, and eco-friendly sensor designs for metal ion detection.
Detecting metal ions at low concentrations poses a considerable challenge.Traditional methods like Atomic Absorption Spectroscopy (AAS), 47,48 Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), 49 and electrochemical assays like voltammetry, 50 are not only time-consuming but also demand expensive equipment and meticulous sample preparation.Consequently, straightforward uorescence probes have surfaced as encouraging sensors.Their attributes, such as high quantum yield, ease of modication, excellent biocompatibility, heightened sensitivity, and the ability to provide real-time sensing. 51,52][55] In this study, we used whey extracted from yogurt as the main material for synthesizing carbon quantum dots (CDs) with a distinct uorescent emission peak at 423 nm.The uorescence at 423 nm, displayed by the CDs, can be quenched by Fe 3+ ions.Leveraging the strong binding affinity between CDs and Fe 3+ ions, we can explore the application of these CDs for detecting Fe 3+ ions in both serum and tap water.

Apparatus
A Cary 60 spectrophotometer from Agilent Technologies, USA, was used to obtain the UV-vis absorbance spectrum (200-600 nm), while a Cary Eclipse Fluorescence spectrophotometer, also from Agilent Technologies, USA, was employed for spectrouorometric scanning.The FEI Tecnai G2 F30 was used for High-Resolution Transmission Electron Microscopy (HRTEM), and a Nicolet FTIR spectrometer from Thermo Scientic, USA, was utilized.X-ray diffraction (XRD) analysis was conducted using an Empyrean XRD with a Cu Ka radiation source.

Synthesis of carbon dots
Yogurt was obtained aer purchasing at a local supermarket in Chamchamal, Iraq.The whey was then collected from the yogurt and utilized for the fabrication of C-dots through a onestep hydrothermal process.The solution was centrifuged at 4500 rpm for 15 minutes to remove impurities, and the resulting supernatant was poured into a Teon-lined stainless-steel chamber.It was then heated at 180 °C for 8 hours in an oven.The resultant solution underwent another round of centrifugation (12 000 rpm for 10 minutes) and was ltered through a sterile PTFE syringe lter (0.22 mm).To eliminate any residual unreacted species molecules, an extraction process was conducted twice, using chloroform as a solvent.The product was collected and refrigerated for further experiments.The process of preparing the luminescent CDs from whey is outlined below and illustrated in Scheme 1.

Analytical application
For the detection of Fe 3+ ions, 0.5 mL of puried CD solution was mixed with varying amounts of ferric nitrate solution.The mixture was then diluted with deionized water to a total volume of 4 mL.Subsequently, the uorescence spectra of the solution were recorded and utilized for quantitative analysis.

Detection of Fe 3+ in serum and tap water
The initial step involved treating human serum to liberate Fe 3+ from proteins.Essentially, equal amounts of human serum and pure ethanol were combined and heated at 95 °C for 15 minutes.Following cooling to room temperature, the serum solution underwent sonication for 2 minutes.Subsequently, the Paper RSC Advances centrifugation process was employed to eliminate the protein precipitate, and the resulting supernatant was gathered for subsequent use. 56All experiments involving human blood serum samples were performed in compliance with the relevant laws and guidelines of Iraq.The experiments followed the institutional guidelines of Chamchamal Sub DOH Central Public Health Laboratory and were approved by its Ethical Committee (Approval No. CPHC100).Informed consent was obtained from all human subjects who participated in the study.In assessing the practicality of the carbon dots-based sensor for detecting Fe 3+ in authentic samples, tap water samples sourced from our laboratory were subjected to analysis using the described method.All the samples of the water were spiked with Fe 3+ at varying concentrations without undergoing any pre-treatment.
3 Results and discussion

Characterization
The structure and dimensions of the synthesized CDs were examined using HRTEM, as depicted in Fig. 1.Fig. 1A and B illustrate that the CDs exhibit uniform spherical shapes and are evenly dispersed.The size distribution, presented in the histogram (Fig. 1C), indicates a range from 1 to 5 nm, with a mean particle diameter of roughly 3.12 nm.Furthermore, the lattice fringes observed in the HRTEM image (Fig. 1C) have a d-spacing of approximately 0.34 nm, consistent with the lattice of graphitic carbon's (100) plane. 57,58Interestingly, the CDs do not exhibit a distinct crystal lattice, implying an amorphous structural nature.FTIR examination was conducted to investigate the active groups present on the surface of the CDs.In Fig. 1B, the following observations were made: stretching vibrations of O-H and N-H in the region 2900 cm −1 to 3400 cm −1 , 59 an absorption peak at 1676 cm −1 corresponding to the stretching vibration of C]O, 60 the absorption peak at 1521 cm −1 attributed to the stretching vibration of C]C, and the peak at 1398 cm −1 identied as C-N, N-H.Additionally, the peak at 1191 cm −1 was ascribed to the C-O, C-N, and C-S bonds. 61The peak at 1023 cm −1 is assigned to the stretching vibration of S]O. 62he XRD pattern of the CDs displays a range of broad peaks, indicating the presence of highly disordered carbon atoms in the as-prepared CDs (refer to Fig. 2). 63,64The CDs exhibited broad 2q patterns around 22.5 and 41°, attributed to disordered carbon atoms which conrms the dominance of C(002) and C(100) planes associated with hexagonal graphite structure of CDs particles. 65he broad XRD pattern is evidence for the crystalline particles with small dimensions.The particle size distribution (Fig. 1C) supports the XRD approach.The Debye-Scherrer equation (D = Kl/b cos q) establishes the fact that the produced CDs are of small size ascribing to the fact the b-factor in the denominator of the equation is too high for broad peak around 2q = 15-30°and consequently the crystallite size will be very small.

The FL properties of CDs
The optical characteristics of the carbon dots were assessed using UV-vis and uorescence spectroscopy.The UV-vis spectrum, illustrated in Fig. 3, revealed a wide absorption band spanning from 250 to 420 nm.Notably, the absorption bands peaked at 283 and 347 nm, matching the p-p* transition and the n-p* transition ascribed to aromatic sp 2 hybridization and C]O bonds respectively.These peaks are attributed to the existence of carbonyl-based groups on the surface. 35The excitation and emission spectra were analyzed, and the excitation spectra were obtained by monitoring the emission at 423 nm, revealing a strong peak at 350 nm.The dots exhibited blue emission at 423 nm under 350 nm excitation.The visual Scheme 1 Illustration of the process for synthesizing the carbon material.
conrmation of C-dot synthesis was established.As depicted in Fig. 3B, hydrothermal synthesis produces a colorless solution (in daylight) that transforms into a blue color under UV excitation (360 nm) of the aromatic sp 2 domains.

The stability experiment
The durability of photoluminescence exhibited by CDs in aqueous settings is a subject of signicant attention for realistic claims.Various factors, such as pH and ionic strength, can inuence their recital.To explore this, the study initially examined the uorescence emission intensity of CDs in solutions containing salt and in different aqueous environments at varying pH levels.The strength of the emission experienced minimal impact across various concentrations of NaCl solutions (Fig. 3C), aligning with ndings from a prior study.Fig. 3D illustrates how the uorescence intensity of CDs is inuenced by pH.The CDs synthesized exhibit stability within the pH range of 2.0 to 12.0, rendering them suitable for a variety of applications.
The stability analysis of CDs reveals no signicant change in uorescence intensity during the three month storage period, as depicted in Fig. 4A.This result suggests that the CDs remained stable even aer three months, with minimal variation.
The uorescence spectra of the C-dots solution, prepared under normal temperature conditions, were recorded.When excited at varying wavelengths spanning from 300 nm to 420 nm with a 10 nm increment, as shown in Fig. 4B, the FL intensity of emission peaks initially increased under excitation from 300 to 350 nm.Subsequently, aer excitation at 350 nm, it gradually decreased.These ndings indicate that the optimal emission was observed at 423 nm when excited at 350 nm.

Selectivity studies
Various metal ions and molecules may exist in the real solution.
The inuence of 20 kinds of metal ions and molecules on the uorescence intensity is compared.The procedure involved creating stock solutions of metals and molecules (0.01 M) and adding 500 mL of the probe to a total volume of 4 mL, resulting in a nal concentration of interferences (1 mM).The uorescence intensity of the mixtures was then recorded at an excitation wavelength of 350 nm.It was found that only Fe 3+ ions can selectively quench the uorescence of synthetic materials, as shown in Fig. 5. Metal ions, including Fe 3+ , Al 3+ , Co 2+ , Cu 2+ , Hg 2+ , Mn 2+ , Mg 2+ , Ni 2+ , Zn 2+ , Pb 2+ , and Cd 2+ , along with molecules such as citric acid (CA), arginine (Arg), alanine (Ala), L- aspartic acid (L-AA), glycine (Gly), ascorbic acid (AA), histidine (His), phenylalanine (Phy), and cysteine (Cys), were chosen to investigate their inuence on C-dots.

Analytical application
To conduct a quantitative investigation and set up calibration curves, a series of standard Fe 3+ solutions were examined.Following optimized circumstances; the calibration plot was generated by drawing uorescence intensity against concentrations of standard Fe 3+ , which is determined by the different degrees of the inter-lter effect, as depicted in Fig. 7. 66 As seen Fig. 2 The XRD spectra of the prepared CDs. in Fig. 6A, the uorescence intensity exhibited a decrease as the Fe 3+ concentration increased from 10 mM to 180 mM.simultaneously, the uorescence emission peak at 423 nm lowered.Fig. 6B describes the relationship between Fe 3+ ion concentration and FL intensity in the range from 10 to 180 mM, in accordance with the following equation: The correlation coefficient is 0.992, the calculated detection limit for Fe 3+ ions is 0.409 mM (S/N = 3) and the limit of quantication is 1.365 mM.
CDs have high sensitivity compared with different probes that have been used to quantication of Fe 3+ in drinking water as shown in Table 1.

Detection of Fe 3+ in real samples
To assess the applicability of this developed technique, standard recovery experiments were carried out using samples of human serum and tap water (refer to Table 2).The results in Table 2 indicate that the recoveries for all samples ranged from 95.60% to 102.46% for human blood serum and from 83.75% to 90.26% for tap water.The associated relative standard deviations (RSDs) were within the ranges of 0.96% to 2.66%.These ndings provide robust evidence supporting the potential of the proposed approach for the practical detection of Fe 3+ in actual samples.

Greenness prole of the method
Recently, energy, waste, and hazard management have been recognized as crucial to improving green analysis.In this work,  three different green metrics were utilized to evaluate the greenness of the proposed approach, comprising the analytical eco-scale (AES), 76 the complimentary green analytical procedure index (ComplexGAPI), 77 and the Analytical Greenness approach (AGREE). 78AES is a simple and semi-quantitative tool that is based on penalty points (PP) for different experimental steps subtracted from a total score of 100.The PPs are assigned for each step of analytical parameters such as the nature and quantity of solvents and reagents employed, energy consumption, hazards, and waste generated.From the results listed in Table 3, one can observe that the proposed method was given a score of 82 for tap water and 78 for blood serum samples, which conrms the greenness of the approach.
The ComplexGAPI metrics soware is an innovative tool built upon GAPI, featuring an additional hexagonal area at its base.ComplexGAPI is essential for encompassing the entire  experimental process, including pre-analysis procedures spanning from sample preparation to the nal analysis.It covers various aspects such as yield and environmental factors, association with the green economy, workup, instrumentation, reagents and solvents, and purication.As in GAPI, the color scale of the pictogram changes from green to yellow to red with respect to the low, medium, and high environmental effects.However, the overall results of ComplexGAPI, as shown in Fig. 7, indicate the greenness of the method.In the suggested procedure, micro-scale extraction was applied as a pre-treatment step for protein precipitation in the blood serum samples to improve the sensitivity of the analysis.Due to the minimal quantity of reagent and solvent waste generated, the procedure seems to be eco-friendly.Whey was utilized as an environmentally friendly carbon source in CD production.Furthermore, the spectrouorimeter consumes little energy per analysis.AGREE Metrics is a soware application that depends on a set of 12 criteria to evaluate the environmental and occupational risks involved in an analytical procedure.These criteria are arranged in a circular pattern resembling the face of a traditional clock, with scores ranging from 0 to 1.The average score, situated at the center, serves as an indicator of the environmental sustainability of the approach.As illustrated in Fig. 8, the high score obtained with the AGREE soware reects the adoption of the excellent green methodology.

Conclusion
Using yogurt whey as a precursor, carbon dots were produced in a one-step hydrothermal method.When stimulated with the maximal excitation wavelength of 350 nm, the produced CDs emit blue uorescent colors at 423 nm.The analytical gure of merit of CDs was investigated, and it demonstrates great selectivity and sensitivity with a low limit of detection for detecting Fe 3+ ions in various matrixes.The addition of ferric ions may nearly completely suppress the uorescence of carbon dots via a uorescence "on/ off" process.Furthermore, by analyzing multiple criteria for the method's greenness, we propose that these carbon dots, due to their greater environmental friendliness, might be used to monitor Fe 3+ ions in a range of biological settings and for environmental monitoring.

Fig. 1 (
Fig. 1 (A) and (B) The HRTEM image of CDs, (C) the statistical distribution of particle sizes of CDs, (D) the pattern of FTIR spectra of CDs.

Fig. 4 (
Fig. 4 (A) Effect of time on the intensity of the CDs.(B) Study of excitation spectra (from 300 to 420 nm) of CDs.

Fig. 5
Fig. 5 Interferences: (A) naked-eye detection of metal ions and molecules.(B) Fluorescence intensity of CD in the presence of 500 mM of various metal ions and molecules.

Fig. 6 (
Fig. 6 (A) The fluorescence behavior of CDs in the existence of varying concentrations of Fe 3+ was examined.(B) Corresponding relationship between FL intensity and the concentrations of Fe 3+ in mM.

Fig. 7
Fig. 7 Schematic of the CDs as an on-off' fluorescent sensor for the detection of Fe 3+ .

Fig. 8
Fig. 8 Evaluation of the environmental friendliness of the proposed method using ComplexGAPI (A) and AGREE (B) scales in tap water (1) and blood serum samples (2).

Table 1
Comparison of other CDs as probes for fluorescence Fe 3+ detection in real samples

Table 2
Recovery results and spiking recoveries of Fe 3+ in human blood serum, and water (n = 3)

Table 3
Greenness evaluation of the proposed method according to the AES a a PPs: penalty points.The assessment was conducted on a per-sample basis.Scores exceeding 75 are classied as excellent green analysis, scores within the range of 50-75 are considered acceptable green analysis, and scores below 50 are deemed inadequate green analysis.